Over the past few years, mass spectrometry, and especially matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been proved to be a vary valuable tool for the structural analysis and characterization of biological macromolecules, such as polypeptides, nucleic acids and oligosaccharides. Nevertheless, application of mass spectrometry, especially MALDI-MS, to the structure analysis of oligosaccharides presents greater challenges than the analysis of polypeptides. The difficulty originates from the intrinsic complexity of oligosaccharide structures and the generally poor response of carbohydrates to standard MALDI, which lead to low sensitivity of the measurement.
During positive ion MALDI, a sample molecule is usually ionized as a protonated ion. Basic groups (such as the side chains of lysine, arginine and histidine in peptides) of the analyte facilitate ionization processes through their strong proton affinity. However, the neutral and acidic oligosaccharides lack basic groups and are usually ionized with lower efficiency by alkali metal cationization when small amount of salts are present). The resulting low sensitivity of oligosaccharides has heretofore limited the application of MALDI for the structure analysis of oligosaccharides.
A variety of derivatization methods for oligosaccharides have been developed to increase the sensitivity of mass spectrometric analysis. Most of these methods use reductive amination. Such procedures usually involve the formation of a covalent linkage by the reduction of a Schiff's base adduct between a primary amine and an acyclic aldehyde or ketone form of the oligosaccharide. This type of derivatization can provide a basic moiety for protonization during ionization, but often result in incomplete reactions, and consequently a multiplicity of products.
Accordingly, it is a principal object of the present invention to provide a method for derivatization of oligosaccharides to facilitate their structure analysis by MALDI.
It is a further object of the present invention to provide a method as aforesaid which is characterized by the derivatization of an oligosaccharide by efficient ligation to a basic aminooxyacetyl peptide by oxime formation reaction, resulting in the formation of a glycoconjugate which yields higher sensitivities in MALDI-MS than the corresponding underivatized oligosaccharide.
It is a further object of the present invention to provide a glycoconjugate derivative of an oligosaccharide which affords an efficient mechanism for the mass determination of oligosaccharides (using mass spectrometry, especially MALDI-TOF MS or ESI MS), as well as fragmentation information from collision-induced dissociation (CID) experiments in the mass spectrometry, especially the MALDI-ITMS instrument.
It is a yet further object of the present invention to provide a method by which digestion of the glycoconjugate derivative by glycosidase arrays in combination with mass spectrometric assay of the digests present a sensitive and rapid approach to elucidation of oligosaccharide linkages, sequence and branching.
It is a still further object of the present invention to provide a faster method than the column chromatography or gel-electrophoresis currently used in commercial oligosaccharide analysis which method can be utilized with impure oligosaccharides, due to the higher resolution of MALDI-MS over column chromatography or gel-electrophoresis.
It is yet another object of this invention to provide a method wherein incomplete glycosidase digestion is acceptable since the structure information of an oligosaccharide is obtained from molecular weight difference, specificities and composition of glycosidase mixture.
It is a further object of the present invention to provide a method of analysis for oligosaccharides wherein the sensitivities is very high, even with the use of picomolar quantities of sample.
It is a still further object of the present invention to provide a method of synthesizing glycoproteins using the novel reaction sequence of the instant invention.
Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description which proceeds with reference to the following illustrative drawings.